Craniofacial malformations comprise one-third of all birth defects, often requiring multiple rounds of invasive surgeries to repair the malformations. Primary cilia are ubiquitous, microtubule-based organelles that coordinate multiple signals critical for the development of the craniofacial complex, including Hedgehog (Hh), Wnt and PDGF signals. Ciliary dysfunction causes a range of clinical disorders, collectively known as ciliopathies, many of which present with severe craniofacial defects. We recently identified a complex of proteins that localizes to the region of the primary cilium between the basal body (ciliary base) and axoneme (ciliary core), termed the transition zone (TZ). This complex regulates intercellular signaling by regulating the ciliary access of membrane-bound receptors and downstream signaling components. Genes mutated in multiple craniofacial ciliopathies, including Meckel and Joubert syndromes, encode proteins of this TZ complex. The underlying molecular mechanism for how disruption of this complex results in craniofacial dysmorphology is currently poorly understood. The goal of this work is to uncover the molecular mechanism by which the TZ regulates normal facial development and how its disruption leads to the defects seen in these ciliopathies. We hypothesize that the TZ complex is a mediator of signaling cross-talk between the developing forebrain and facial signaling center to direct the migration and fusion of the facial primordia during development. Consistent with this hypothesis, we have found that loss-of-function mutations in TZ components causes defects in neural tube patterning and decreased expression of Hh in the facial signaling center. This resulted in severe narrowing of the facial midline (hypotelorism). In this proposal we will use advanced geometric morphometrics to provide comprehensive quantitative analysis of the facial defects in multiple TZ mutants and correlate this with defects in intercellular signaling. Further mechanistic analysis will determine if neural crest specification/migration defects underlie the craniofacial abnormalities in these TZ mutants. Results from this study will aid in our understanding of how primary cilia regulate facial development and may help in the development of new, innovative approaches to treat or prevent craniofacial defects.